Extracellular matrix permeability/efficacy assay tip (E-PAT) to realize three-dimensional cell-based screening

Abstract In this work, an extracellular matrix permeability/efficacy assay tip (E-PAT), which can be used in a conventional pipette to simultaneously test drug permeability and efficacy, was developed by growing cells in the extracellular matrix (ECM) layer in a hole at the outlet (bottom) of E-PAT. In the existing permeability/efficacy assay technique conducted using the transwell assays, the Cell/ECM layers were formed on permeable polymer membranes; however, the production of small membranes to realize transwell miniaturization is expensive and difficult. To resolve this issue, in the proposed approach using E-PAT, small Cell/ECM layers were formed in the hole at the outlet (bottom) of the tip without membranes. A pipette was connected to the E-PAT to easily aspirate the Cell/ECM mixture into the hole of the tip, allowing the formation of miniature Cell/ECM layers at the bottom of the tip. Subsequently, the E-PAT was detached from the pipette and dipped into a well filled with media. The cells in the ECM grew three-dimensionally and made Cell/ECM layer in the E-PAT. The permeability/efficacy assays were performed using the Cell/ECM layer in E-PAT. The compounds or nanoparticles supplied through the inlet (hole) at the top of the E-PAT penetrated the Cell/ECM layers and were diluted by the media in the well. By measuring the concentration of the compounds or nanoparticles in the well and the cell viability in the Cell/ECM layer, the permeability and efficacy/cytotoxicity of the compounds in the proposed E-PAT could be concurrently estimated with a high throughput. In particular, E-PAT measured the IC50 (half maximal inhibitory concentration) for 3.383 µM doxorubicin (DOX) and indicated that 73% 4 µM DOX penetrated the Cell/ECM layers, inducing the death of 57.2% HepG2 cells. Moreover, the device demonstrated that SiO2 nanoparticles with a diameter of 25 nm permeated higher into the ECM and killed more HepG2 cells under the FBS (fetal bovine serum)-free media conditions than under the FBS-containing media conditions. The E-PAT could thus measure the compound efficacy as well as the compound permeability. The proposed tip can be a valuable tool to determine whether the efficacy/cytotoxicity of a compound is because of its low permeability.